A classic field of application of light microscopy for examining biological preparations is that of luminescence microscopy. This involves the use of certain dyes (known as phosphores or fluorophores) for specific marking of samples, e.g. of cell components. As mentioned above, the sample is illuminated with exciting radiation and the luminescence light excited thereby is detected by suitable detectors. For this purpose, a dichroic beam splitter is usually provided in the light microscope in combination with block filters which split off fluorescence radiation from the exciting radiation and enable separate observation. This approach enables the imaging of individual, differently dyed cell components through the light microscope. Of course, it is also possible to dye several portions of a preparation simultaneously with different dyes specifically depositing on different structures of the preparation. This method is referred to as multiple luminescence. It is also possible to measure samples which luminesce per se, i.e. without addition of dyes.
As is common practice, luminescence is understood herein to be the general term for phosphorescence and fluorescence, thus covering both processes.
It is further known in the examination of samples to use laser scanning microscopes (also abbreviated as LSM) which, by means of a confocal detection arrangement (this is then referred to as confocal LSM) or by non-linear sample interaction (known as multi-photon microscopy), image only that plane of a three-dimensionally illuminated image which is located in the focal plane of the objective. An optical cut is obtained, and recording a plurality of optical cuts at different depths of the sample subsequently allows to generate a three-dimensional image of the sample with the help of a suitable data processing device, which image is composed of the various optical cuts. Thus, laser scanning microscopy is suitable to examine thick preparations.
Of course, use is also made of a combination of luminescence microscopy and laser scanning microscopy, wherein a luminescent sample is imaged at various depth levels with the help of an LSM.
Special illumination configurations, such as e.g. a 4Pi-arrangement or arrangements with standing-wave fields, are known for optimal resolution within these limits. Thus, the resolution, in particular in an axial direction, can be considerably improved over a classic LSM. Further, the resolution can be increased to a factor of up to 10 with respect to a diffraction-limited, confocal LSM with the help of non-linear depopulating processes.
FIGS. 1a/b show such a method as described, for example, in U.S. Pat. No. 5,866,911. For resolution enhancement, light radiation having two wavelengths is used. The light radiation of one wavelength is focused onto the sample to be measured as an exciting light beam 1 by means of an objective and excites luminescence, in this case fluorescence, in the sample. For simplification, the representation of FIGS. 1a/b only shows the one-dimensional case. The enhancement in spatial resolution is then effected in that a light beam 2 having a different wavelength depopulates partial areas of the fluorescent state excited by the exciting light beam. Therefore, this light beam is also referred to as “quenching radiation”. For example, irradiation is then effected such that the main maximum of the quenching light beam 2 and the main maximum of the exciting light beam 1 partially overlap, as is clearly recognizable in FIG. 1a. Due to this “depletion” of the sample at the edges of the area illuminated by exciting radiation 1, only a reduced volume 3 still emits fluorescence, as is clearly visible in FIG. 1b. As a consequence, resolution is enhanced due to this reduction in volume.
FIGS. 2a-c show three possible mechanisms using which such depopulation can be effected. FIG. 2a shows the process of stimulated emission depletion (STED). The exciting radiation is applied to the flourophore of state S1 (arrow A). The depopulation of the thus excited state S1 is effected in the direction of the basic state S0 by light radiation having a wavelength in the range of the fluorescence wavelength. The arrow SE shows this stimulated emission whose wavelength is almost identical with that of the luminescence (arrow F). Thus, the exciting wavelength has a wavelength shorter than the depopulating quenching radiation by the amount of the Stokes shift. Thus, the resolution enhancement according to this approach requires two different sources of light, which is also supported by the prior art constituted by DE 4416558 C2.
FIG. 2b illustrates a further possible process of depopulation for the excited state S1 (arrow A) by effecting excitation up to a still higher state S2 (arrow A+) which can no longer emit luminescence. Such raising is referred to as Excited State Absorption, which is why this approach is also abbreviated as ESA. A corresponding description of this process is found, for example, in U.S. Pat. No. 6,633,432. Since the distances of the energy states in a sample or in a dye, respectively, decrease at higher states, the ESA process uses a light source for depopulation which has less energy and, thus, a longer wavelength than that used for excitation. Accordingly, two different light sources are required again.
A further method of depopulation in the case of fluorescence is known as Reversible Saturable Optical Fluorescence Transition, which is described e.g. in DE 10 325 460 A1 and illustrated in FIG. 2c. For imaging at a high spatial resolution, this approach uses a dye which can be repeatedly switched from a first state 5, in which fluorescence occurs, to a second state 6, in which the dye does not fluoresce, with the help of a switching beam 4, said dye being able to return from the second state 6 to the first state 5, as illustrated in FIG. 2c. Partial areas of the sample with the dye are switched into the second state 6 by the switching beam 4, leaving out a defined area of the sample. Fluorescence light 7 is then excited by an exciting beam 1 and is subsequently recorded. The fluorescence light 7 then only comes from sample volumes which have not been previously irradiated with the switching beam 7. By a suitable overlap of the exciting beam 1 and the switching beam 4, the volume from which the fluorescence light 7 is emitted is smaller than obtainable a priori through the resolution of the exciting beam 1 and the sharpness of the zero of the switching beam 4.
Thus, in all of the three cited methods according to FIGS. 2a to 2c fluorescence is prevented by the use of light radiation having a wavelength not equal to the wavelength for excitation. At the same time, this light radiation has to comprise at least one distinctly limited, local zero of radiation power, which determines the final resolution of the detected fluorescence radiation. When the zero is only provided as a minimum and does not completely vanish, the fluorescence power and, thus, the efficiency of the method will decrease further. This is the case, for example, where aberrations occur in the optical arrangement or in the preparation, respectively.
FIG. 3 shows a known device using one of the three aforementioned methods for resolution enhancement, which is the STED process of the example of FIG. 3. An excitation radiation source 8 generates an Airy distribution in the sample 10, by which the sample is brought from the ground state S0 to the excited state S1. The depopulation of the state S1 is effected by means of a quenching light source 11 which, when a phase plate 12 is used, has a donut-shaped or toroidal spectral composition 13 in the sample 10. The luminescence radiation of the un-depopulated, i.e. undepleted, dye molecules is detected with the help of a detector 14. The depopulation causes the resolution of the microscope to be enhanced beyond the diffraction limit resulting from the Airy distribution. This is shown by a reduced point spread distribution 15 of the high resolution microscope as compared to the conventional microscope 16.
A further method of resolution enhancement is addressed in EP 1 157 297 B1. In said method, non-linear processes are to be utilized by means of structured illumination. As non-linearity, the document mentions the saturation of fluorescence. The described method claims to realize a shift of the object space spectrum relative to the transfer function of the optical system by structured illumination. More specifically, the spectral shift means that object space frequencies V0 are transmitted at a space frequency of V0-Vm, with Vm being the frequency of the structured illumination. At a given space frequency which is the maximum frequency transmissible by the system, this enables the transfer of space frequencies of the object which exceed the maximum frequency of the transfer function by the shifting frequency Vm. This approach requires a reconstruction algorithm for imaging and the evaluation of several photographs for one image.